Ion exchange, chromatographic separation columns, and immobilized enzyme reactors typically use a column packed with a suitable support material. In many separation or purification techniques, enzymes or other proteins are adsorbed to the outer surfaces of the support material or "carrier". The reaction solution is usually fed into the top of the reaction column (often under pressure) and is eluted after passing through the reaction column.
Cellulose-based resins are sometimes used as support material or carriers for immobilized enzymes or other charged substances including yeast cells or bacteria. The support material is typically in the form of irregularly shaped particles or "beads". Methods for the preparation of cellulose beads including dispersion of viscose cellulose into a suitable solvent are disclosed in U.S. Pat. No. 3,597,350 to Determan; Great Britain Patent No. 1,458,955; Swedish Patent No. 7,505,610 to Peska et al.; U.S. Pat. No. 4,055,510 to Peska; DE Patent Nos. 2,714,965 to Tolsdorf and 2,523,893 to Walser and DDR Patent No. 147,114 to Gensrich et al. In addition, cellulose beads can be prepared by dispersion in water (U.S. Pat. No. 4,063,017 to Tsao et al. and U.S. Pat. No. 4,090,022 to Tsao et al.), extrusion of cellulose together with a polymer (U.S. Pat. No. 3,501,419 to Bridgeford) and agglomerization of cellulose fiber with a resin as disclosed in U.S. Pat. Nos. 4,110,164 to Sutthuff and 4,355,117 to Antrim et al.
A cellulose-based carrier material can, for example, be prepared by admixing a polymer (for example, polystyrene), a densifier and fibrous cellulose, and melt extruding and grinding the admixture into particles to create irregularly shaped beads. In addition, a derivatization step is usually carried out to prepare diethylaminoethyl (DEAE) cellulose so that such particles, when exposed to an aqueous enzyme solution, are capable of binding the protein.
Although particles generated by these methods are generally useful, they have certain drawbacks. The cellulose, which binds the proteins exposed to the support material, is not uniformly distributed on the outer portions of the support material; the cellulose is generally incorporated into the matrix of the particle. As a result, much of the cellulose utilized is not available for protein binding. The cellulose which is a part of the particle is thus not maximally utilized and the overall absorption capacity is not what it theoretically could be if all, or substantially all of the cellulose is available for binding.
In addition, particles generated by these methods, particularly extrusion and grinding, agglomerization, and dispersion techniques, are irregularly shaped; these irregularly shaped particles can create serious "channeling" problems during continuous or semi-continuous column processes, which reduces the effectiveness and efficiency of the chromatographic separation, ion exchange, and conversion by immobilized enzyme processes. Cellulose based beads are often relatively soft and tend to shrink and swell--depending on the moisture and salt concentration in the column--which also reduces the effectiveness of the process. Softer materials cannot effectively be used with high flow rates and elevated pressures.
The present invention discloses adsorption material which has increased binding capacity of proteins and other charged particles, more efficient utilization of cellulose, a lesser tendency to shrink and swell and, in the form of substantially spherical particles, has improved packing and operational capabilities. The present invention contemplates a support particle which has cellulose capable of binding charged molecules such as proteins, distributed over substantially all of the exterior surfaces thereof. Rather than distributing the cellulose throughout the matrix, the present invention provides for particles with cellulose distributed on the exterior surfaces substantially all of which is available for binding charged molecules such as proteins. Hence, the material of the instant invention requires less cellulose, but will exhibit a higher binding capacity which results in increased effectiveness of the column due to the higher enzyme or other protein activity present. In addition, unlike softer cellulose based material, the material of the instant invention is not affected by the "wetness" of the reaction vessel and other conditions and will not shrink or swell.
It is known that in certain contexts, irregularly shaped particles create channeling and other problems. Spherical material tends to reduce or eliminate these problems. Generation of cellulose based spherical particles on an efficient and practical scale is not disclosed by the prior art.
In one specific aspect, the present invention discloses substantially spherical adsorption material which comprises support particles which have cellulose distributed over substantially all of the exterior surfaces thereof. Spherical support material reduces or eliminates "channeling" in the reaction vessels.
The instant invention also discloses a method for generating substantially spherical particles by the use of "prilling", i.e. dispersing droplets of a hot melt at the top of a prilling tower which is of sufficient height to permit the droplets to cool and form relatively spherical particles before they reach the bottom of the tower. A mixture of cellulose with support material such as wax matrices and/or polymers is too viscous to effectively "prill" in this fashion. According to the method of the present invention, spherical support particles of sufficient density are generated by "prilling" and cellulose is then distributed over the exterior surfaces of the support particles by, for example, standard fluidized bed techniques. The resulting material is well suited for use in ion-exchange chromatography, enzyme immobilization or other separation or purification expedients.